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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
not specified
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 20 June 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at
Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Preparation of Inoculum
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that
may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.
Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (50 mL) of the washed activated sewage sludge by suction through preweighed
GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized
reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a
constant weight was attained. The suspended solids concentration was equal to 3.2 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The test item was dissolved directly in mineral medium. A nominal amount of test item (500 mg) was dissolved in mineral medium and the volume adjusted to 1 liter to give a 500 mg/L stock solution. An aliquot (114 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 19.0 mg/L, equivalent to 10 mg carbon/L. The volumetric flask
containing the test item was inverted several times to ensure homogeneity of the solution.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 22 and 25 °C, in darkness. On Day 18 of the test the temperature in a vessel containing water which was incubated under the same conditions as the test vessels was recorded as being 24.5 °C. This was a deviation from the Study Plan which states the test will be conducted at a temperature of 22 ± 2 ºC. This deviation was considered to have not affected the integrity or validity of the study given that all validation criterion were met.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 34.6 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. If necessary the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.
The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.

Observations
The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

pH Measurements
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge . Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.
Test performance:
Definitive Test
Percentage biodegradation values of the test and reference items and the toxicity control biodegradation curves are presented in Figure 1 and in the results table..

Validation Criteria
The total CO2 evolution in the inoculum control vessels on Day 28 was 34.20 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion
given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test
Guidelines.

Biodegradation
The test item attained 88% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the
biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 70% biodegradation after 14 days and 73% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the
sewage treatment micro-organisms used in the test.
Sodium benzoate attained 72% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
0 d
Parameter:
% degradation (CO2 evolution)
Value:
31
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
70
Sampling time:
6 d
Parameter:
% degradation (CO2 evolution)
Value:
79
Sampling time:
8 d
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
78
Sampling time:
14 d
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
21 d
Parameter:
% degradation (CO2 evolution)
Value:
88
Sampling time:
28 d
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
6-chloro-1hexanol attained 88% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the
biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Executive summary:

6-chloro-1hexanol attained 88% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

6-chloro-1hexanol attained 88% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information